Summary of Work: Genetic defects as well potentially unstable at-risk DNA motifs (ARMs) can cause genome instability and the combination can lead to synergistic increases in instability and disease in humans. Yeast provides an in vivo test tube for functional analysis of human DNA metabolic genes and ARMs. We used ARMs to address genome stability and to detect subtle defects in DNA metabolic genes, reasoning that variants with a small effect might exhibit strong synergistic interactions between alleles, or with ARMS or with environmental factors. A) Alterations in the essential DNA polymerase e impact on genome stability. An allele was isolated that specifically increases +1 frameshift mutations in long homonucleotide runs by lowering DNA replication fidelity. In combination with a proofreading defect the double mutant is the strongest polymerase e mutator identified and is lethal in a mismatch repair background. B) The 5' DNA flap endonuclease hFEN1, which is important for human replication and repair, could fully complement a yeast null RAD27 mutant. The several genetic effects of a nuclease-deficient allele led to the isolation of novel genotoxic hFEN1 mutants. A mutant RAD27/FEN1 that lacks interaction with PCNA appears to have little effect on genome stability; however, it exhibited synergy with double-strand break (DSB) repair mutants. C) We discovered (through an IRA collaboration with the Kunkel lab) strong negative interactions between a subtle allele of RAD27/FEN1 and defects in the DNA polymerase d 3'->5' exonuclease (but not in other domains of Pol d) that caused hyper mutation and recombination, DSBs and even cell death. This demonstrates a novel role for this exonuclease in addition to proofreading. D) Many combinations of subtle mutator alleles of DNA polymerase d combined with strong recessive mutators can result in a strong mutator phenotype in diploid cells. E) collaborating with the Wilson lab we developed a yeast system to investigate human DNA polymerase b, which can play a major role in the repair of DNA damage.